During the past few decades there has been an alarming rise in the prevalence of chronic kidney disease (CKD) worldwide. Atherosclerotic cardiovascular disease is the major source of mortality in CKD patients. Decreased HDL levels and defective HDL-mediated reverse lipid transport are major contributors to the atherogenic diathesis in the CKD population. A major cause of decreased serum HDL level in CKD is the reduction in apoliprotein A-l, the major structural and functional component of HDL. Animal studies in our lab have shown decreased ApoA-l in serum and decreased mRNA expression in liver of uremic rats. Furthermore, decreased ApoA-l expression, synthesis and secretion by human liver cells under uremic conditions has been documented. Given the preponderance of evidence linking ApoA-l deficiency to increased risks of cardiovascular events and mortality, studies aimed at deciphering the mechanisms responsible for the ApoA-l deficiency of CKD are of vital importance. We hypothesize that uremic toxins present in the serum of CKD patients are responsible for down regulation of ApoA-l at the transcriptional level. Using an ApoA-l promoter-luciferase construct, we have shown decreased ApoA-l promoter activity in liver cells exposed to uremia. Furthermore, we believe this inhibitory effect is mediated through a uremia responsive element (URE) within the ApoA-l promoter. Therefore, binding of one or more transcription factors to URE in the promoter of the ApoA-l gene results in decreased transcriptional activity. In addition, we hypothesize that the effect of uremia on the URE occurs in both the liver and intestinal cells (the two cell types responsible for producing serum ApoA-l). Using deletion mapping and a promoter luciferase reporter construct, we hope to identify a URE in the ApoA-l promoter. Subsequently, site-directed mutagenesis will be utilized to identify cis-regulatory sites within the URE. Furthermore, transcription factors potentially involved in ApoA-l regulation will be identified using computational analysis. Finally, using electrophoretic mobility shift and supershift assays, binding of the identified regulatory proteins to the cis-regulatory elements in the ApoA-l promoter will be confirmed. These studies will be carried out in human liver (HepG2) and intestinal (Caco2) cells. The epidemiological observation that decreased ApoA-l is associated with increased cardiovascular mortality along with the fact that CKD is a state of ApoA-l deficiency makes this molecule an attractive target for therapy. Once the mechanisms responsible for ApoA-l deficiency in CKD have been identified, then more effective therapies aimed specifically at correcting those deleterious mechanisms can be devised.